Directly injected DNA can express its encoded proteins and elicit specific immune responses in animals (Wolff et al., “Direct Gene Transfer Into Mouse Muscle in vivo,” Science 47:1465–1468 (1990); Donnelly et al., “DNA Vaccines,” Annu. Rev. Immunol. 15:617–648 (1997)). Most of the DNA delivery technologies reported so far have been focused on naked DNA delivery (as in DNA vaccination) and non-viral or viral vector mediated systems (Anderson, “Human Gene Therapy,” Nature 392:25–30 (1998)). Clinical applications of viral-mediated systems have been delayed by safety issues such as mutagenic potential and immunogenicity (Crystal, “Transfer of Genes to Humans: Early Lessons and Obstacles to Success,” Science 270:404–410 (1995) and Tripathy et al., “Immune Responses to Transgene-encoded Proteins Limit the Stability of Gene Expression After Injection of Replication-defective Adenovirus Vectors,” Nat. Med. 2:545–550 (1996)). The generally poor efficiency of delivery and expression by non-viral systems remains one of the main limitations to the development of gene therapy and DNA vaccination (Thierry et al., “Characterization of Liposome-mediated Gene Delivery: Expression, Stability and Pharmacokinetics of Plasmid DNA,” Gene Ther. 4:226–237 (1997) and Liu et al., “Cationic Liposome-mediated Intravenous Gene Delivery,” J. Biol. Chem. 270:24864–24870 (1995)). Much attention is therefore being paid to the design of new formulations of DNA with various substances, such as lipid (Ishii et al., “Cationic Liposomes are a Strong Adjuvant for a DNA Vaccine of Human Immunodeficiency Virus Type 1,” AIDS Res. Hum. Retroviruses 13:1421–1428 (1997)), polycation/polysaccharide (Erbacher et al., “Chitosan-based Vector/DNA Complexes for Gene Delivery: Biophysical Characteristics and Transfection Ability,” Pharm. Res. 15:1332–1339 (1998)), peptide (Erbacher et al., “The Reduction of the Positive Charges of Polylysine by Partial Gluconoylation Increases the Transfection Efficiency of Polylysine/DNA Complexes,” Biochim. Biophys. Acta 1324:27–36 (1997)), peptoid (Murphy et al., “A Combinatorial Approach to the Discovery of Efficient Cationic Peptoid Reagents for Gene Delivery,” Proc. Natl. Acad. Sci. USA 95:1517–1522 (1998)), gold particles (Fynan et al., “DNA Vaccines: Protective Immunizations by Parenteral, Mucosal, and Gene-Gun Inoculations,” Proc. Natl. Acad. Sci. USA 90:11478–11482 (1993)), protein (Hart et al., “Gene Delivery and Expression Mediated by an Integrin-binding Peptide,” Gene Ther. 2:552–554 (1995)), polymers (Katayose et al., “Water-soluble Polyion Complex Associates of DNA and Poly(ethylene glycol)-poly(L-lysine) Block Copolymer,” Bioconjug. Chem. 8:702–707 (1997)), and other complexes (Kim et al., “A New Non-viral DNA Delivery Vector: The Terplex System,” J. Controlled Rel. 53:175–182 (1998)). All of these systems deliver DNA as a bolus, without long-term sustained release.
Controlled release systems using biocompatible and/or biodegradable polymers provide an attractive alternative for long-term delivery of therapeutic agents (including DNA). There are many advantages of polymer-mediated controlled release systems over conventional delivery systems (Mahoney et al., “Controlled Release of Proteins to Tissue Transplants for the Treatment of Neurodegenerative Disorders,” J. Pharm. Sci. 85:1276–1281 (1996)). These include: (i) therapeutic agents can be delivered to tissues in a sustained, continuous and predictable fashion; (ii) therapeutic agents are well protected before being released; (iii) site specific delivery (such as in brain) can be achieved by simple implantation or direct injection; and (iv) repeated drug administration is not necessary. Despite the fact that in recent years controlled release systems have been successfully employed to deliver proteins and other macromolecules (Cohen et al., “Controlled Delivery Systems for Proteins Based on Poly(lactic/glycolic acid) Microspheres,” Pharm. Res. 8:713–720 (1991), Saltzman et al., “Transport Rates of Proteins in Porous Materials with Known Microgeometry,” Biophys. J. 55:163–171 (1989); and Siegel et al., “Controlled Release of Polypeptides and Other Macromolecules,” Pharm. Res. 1:2–10 (1984)), polymer-based DNA controlled release systems have not been fully explored. Several previous reports of DNA delivery using synthetic polymers have important limitations, such as limited range of DNA sizes and DNA dosages, reliance on non-FDA approved materials, difficulty in control of release rate (Mathiowitz et al., “Biologically Erodable Microspheres as Potential Oral Drug Delivery Systems,” Nature 386:410–414 (1997); Jong et al., “Controlled Release of Plasmid DNA,” J. Controlled Rel. 47:123–134 (1997); Labhasetwar et al., “A DNA Controlled-release Coating for Gene Transfer: Transfection in Skeletal and Cardiac Muscle,” J. Pharm. Sci. 87:1347–1350 (1998); Wang et al., “Encapsulation of Plasmid DNA in Biodegradable Poly(D, L-lactic-co-glycolic acid) Microspheres as a Novel Approach for Immunogene Delivery,” J. Controlled Rel. 57:9–18 (1999); and Ando et al., “PLGA Microspheres Containing Plasmid DNA: Preservation of Supercoiled DNA via Cryopreparation and Carbohydrate Stabilization,” J. Pharm. Sci. 88:126–130 (1999)).
The present invention is directed to identifying factors responsible for modifying the controlled release rate of nucleic acid from biocompatible polymers, and otherwise overcoming the above-described deficiencies in the relevant art.